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Where are they now? Featuring: Jay Guillory
?????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? Where are they now?
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Justin “Jay” Guillory
MEd in Educational Leadership, concentration in Student Personnel Leadership
Department of Educational Leadership and Policy Analysis
Year of Graduation: 2020 What is your current position and/or research?
My current position is Interim Assistant Director of the Mary V. Jordan Multicultural Center. My current research interest is the impact of summer bridge programs on underrepresented students’ persistence, retention, and graduation rate.
Why did you choose ETSU for your education?
Originally, I was looking at ETSU for their Master of Arts in History. The staff in the history department are more diverse compared to the department I received my undergraduate degree from. When I finally figured out my career path was student affairs, I begin to look into the student personnel program (now higher education leadership) at ETSU, and it was a great fit for me. What solidified the decision for me was being offered a graduate assistantship in the Mary V. Jordan Multicultural Center by then Director Carshonda Martin. What does this position/research entail?
In this position, I am responsible for the center access programs (B.U.C.S Academy, QUEST for Success, and Discover ETSU) while also being responsible for the leadership development of our student leaders. I also help with the day-to-day operations of the Multicultural Center.
How did your time at ETSU prepare you for this career?
My time at ETSU prepared me for my career because my graduate assistantship advisor allowed me to have a seat at the table when it came to planning programs. Experience with program planning allowed me to be better prepared when taking on my coordinator role and now interim assistant director role.
What advice would you offer current or future graduate students?
The road is sometimes rough. But it’s important to take care of yourself! Self-care is the best care! Also, lean on your cohort members. They are here to support you just as much as faculty members are.
Anything else you would like to add? GO BUCS!
Determining the Molecular Structure of a Grapefruit Enzyme Using X-ray Crystallography
Aaron Birchfield
Biomedical Sciences, PhD Cellular, Molecular, and Chemical Biology Concentration
Dr. Cecilia McIntosh
Faculty Advisor
Written by Marten Baur
“There are tens of thousands of glucose transferring enzymes in plants, and only six have been crystallized,” remarked Dr. Cecilia McIntosh, professor emerita in ETSU’s Department of Biomedical Sciences. Aaron Birchfield, a second year PhD student under Dr. McIntosh, is drawing closer to crystallizing the seventh-ever glucose transferase enzyme found in plants. This enzyme, derived from grapefruit, is called Citrus paradisi 3-glucosyl-transferase (CP3GT), and serves the unique role of attaching a glucose molecule onto the third carbon of a flavonol compound. While CP3GT is important to the grapefruit plant’s survivability, its applications extend well beyond these functions. This article features Aaron’s research and highlights his contributions to the field of biomedical sciences.
Aaron Birchfield
While Aaron never anticipated studying grapefruit, he has always dreamt of becoming a scientist. With an innate curiosity of the natural world, Aaron recalls reading children’s encyclopedias in the second grade. Now, as he develops protein crystals in Dr. McIntosh’s lab, he is well along his way to fulfilling his dream. Aaron grew up in Bristol, Virginia, and he received his bachelor’s degree in chemistry and his master’s degree in biology from ETSU. As a transfer student from King University, Aaron was drawn to ETSU because of its robust chemistry department with an emphasis on research. ETSU’s friendly atmosphere and invested professors, along with opportunities for undergraduate research, motivated Aaron to move to Johnson City to complete his bachelor’s degree. Aaron is fascinated with protein crystals, which are pure proteins that form into crystals under specific conditions. These crystals can be used to determine the three dimensional structure of a protein through a process known as X-ray crystallography. “This idea that you can make crystals to determine—atom per atom—how an enzyme is put together in three-dimensional space just blew my mind!” expressed Aaron.
Aaron is mentoring under Dr. McIntosh, who has studied grapefruit for over forty years and is well-versed in protein and enzyme biochemistry. Dr. McIntosh and Aaron’s research focuses on flavonoid biosynthesis and regulation. Flavonoids are a class of compounds that protect a plant’s photosynthetic machinery from the sun’s ultraviolet rays, aid in the formation of nitrogen-fixing root nodules, and increase plant-pollinator interactions. Importantly to humans, flavonoids contribute to a plant’s taste, smell, and color. Flavonoids are known for their antioxidant properties, and they are marketed as nutritional supplements such as quercetin, kaempferol, and myricetin. In addition to supplements, flavonoids are found in several cancerfighting drugs, and they have even been found to reduce excessive inflammation induced by COVID-19. Aaron and Dr. McIntosh are currently working to identify the molecular structure of CP3GT, an enzyme that attaches glucose to the third carbon on a flavonoid molecule. Because CP3GT has not been crystallized, its exact three-dimensional molecular structure is still unknown.
A compound’s structure determines its function. Therefore, identifying a protein’s exact structure has several practical applications. Synthetic chemists and biologists use enzymes when creating compounds such as medicines or nutraceuticals. Just as a carpenter would use a hammer to pound a nail, synthetic chemists use enzymes as tools to accomplish specific tasks when synthesizing compounds. When an enzyme’s structure has not been determined through protein crystallization, chemists must rely on computer-generated models to approximate the structure and function of the enzyme. Because the models are not 100% accurate, enzymes can act in unanticipated ways. This can make it difficult for synthetic chemists to use the enzyme, as the enzyme might accomplish a different task than expected. Just as a hammer can serve many different functions other than pounding nails, an enzyme in the hands of a skilled scientist can be mutated to
accomplish several similar, but different tasks. By identifying the exact structure of CP3GT, scientists will have another tool in their tool box when synthesizing medicines or supplements.
Aaron and Dr. McIntosh want to know the specific structural features of CP3GT that allow it to attach a glucose to the third carbon position of a flavonol and not to any other types of flavonoid compounds. They hypothesize that if they can identify the structural elements of CP3GT through X-ray crystallography, then they can understand what gives rise to its unique activity. Before beginning his crystallization experiments, Aaron first had to obtain sufficient amounts of CP3GT. Aaron took the DNA responsible for synthesizing CP3GT from grapefruit, added a yeast promoter sequence and histidine tags to the ends of the DNA strand, and inserted it into a yeast cell’s DNA. Yeast cells prefer to eat glucose, but in the presence of methanol, the yeast will turn on a gene to create enzymes that break down methanol. This gene has a promoter sequence identical to the promoter Aaron attached to the inserted CP3GT DNA. Because of this, when the yeast turns on the gene to make the methanol enzymes, it also makes CP3GT. As Dr. McIntosh succinctly described, “We turn yeast into a CP3GT-producing factory.”
The histidine tags Aaron attached to the CP3GT DNA allow him to isolate CP3GT from the other yeast proteins through the process of affinity column chromatography—a method used to separate compounds poured through a vertical glass column.
Because the histidine tags attached to CP3GT have a high affinity for cobalt, Aaron uses a cobalt stationary phase (a gel-like material that the solution passes through to separate the proteins) to catch the CP3GT while the other yeast enzymes flow through the column freely. The process of culturing yeast to produce histidine-tagged CP3GT was a huge accomplishment, and Aaron published a paper based on his work. While difficult, culturing yeast cells was his favorite part of the project. “I’ve probably grown enough yeast to power a small bakery!” joked Aaron.
Aaron was able to isolate 85% pure CP3GT; however, in order to form a crystal, he needs a purity of 95%. Aaron must develop additional purification steps in order to form CP3GT crystals and determine the enzyme’s structure. His next steps are to run an anion exchange, which uses the same column chromatography technique but separates the proteins based on their electric charge rather than their affinity for cobalt. “It takes multiple experiments to figure out what conditions to use and what works,” explained Dr. McIntosh. With luck, Aaron will achieve 95% purity by summer of 2022.
Dr. McIntosh has helped Aaron develop as both a scientist and writer. “Dr. McIntosh played a huge role in getting my writing where it needed to be in order to write and publish papers,” expressed Aaron. He now has two peer-reviewed publications in Reports of Biochemistry and Molecular Biology and Current Plant Biology. After publishing his project on the crystallization and determination of the molecular structure of CP3GT, Aaron will have fulfilled his research requirements for his PhD. Aaron has already presented his research at the Phytochemical Society of North America in 2019 and 2021, and he plans on presenting at the Appalachian Student Research Forum this spring and at PSNA again in summer 2022. After Aaron graduates in summer of 2023, he plans to work in the medical industry. He wants to apply everything he has learned and use it to better human medicine or health: “I really just want to grow someone a new set of lungs!” stated Aaron.
Aaron, a devout Christian, brings a unique perspective to the field of science. He is fascinated by the complex interplay of chemical reactions that produce the experience of life. Aaron explained, “Studying the biochemical foundations of life gives me a window into the eyes of God—how he built the foundations of life and shaped humanity.” Aaron hopes to determine the molecular structure of CP3GT by fall of 2023, and views his work as his own “small contribution to the Creator’s blueprint.” By determining the structure of CP3GT, Aaron and Dr. McIntosh bring humanity a small step closer to understanding the phenomena that shape the natural world.
Left, Dr. Cecilia McIntosh, Right, Aaron Birchfield
Aaron Birchfield birchfieldas@etsu.edu
